KR20140077178A - Separation method for fucosylated antibodies - Google Patents

Abstract

The invention relates to a method of separating antibodies, in particular antibodies having different degrees of fucosylation. The method is based on the binding affinity of the antibody to the Fc receptor. The invention also relates to the use of Fc receptors for the isolation of antibodies with different degrees of fucosylation.

Description

[0001] SEPARATION METHOD FOR FUCOSYLATED ANTIBODIES [0002]

The invention relates to a method of separating antibodies, in particular antibodies having different degrees of fucosylation. The method is based on the binding affinity of the antibody to the Fc receptor. The invention also relates to the use of Fc receptors for the isolation of antibodies with different degrees of fucosylation.

Human IgG ₁ is an invariant Fc (fragmentable fragment) domain that mediates immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and interacts with components of the immune system and And two Fab (fragment antigen binding) fragments including a variable region responsible for antigen recognition. The carbohydrate structure attached to the N-glycosylation site conserved in the asparagine 297 (Asn297, N297) in the CH2 domain of the constant region is essential for mediating these effector functions (1-4).

Naturally, the oligosaccharides attached to the Fc domain are primarily biantennary complex-type structures with varying contents of bisecting GlcNAc (N-acetylglucosamine), terminal galactose, central fucose and sialic acid ).

Recent studies have shown that modification of carbohydrate composition significantly affects antibody-mediated immune effector function (3-5). Low levels of galactosylation positively affect complement activation whereas a lack of central fucose promotes high binding affinity of Fc [gamma] RIIa, thereby enhancing ADCC (5-7). Several approaches have been developed to manipulate glycosylation profiles and produce therapeutic antibodies with improved biological function (8-10).

For example, in mammalian cells overexpressing? (1,4) -N-acetylglucosaminyltransferase (GnT) III and mannosidase (Man) II, glycoengineering antibodies It is characterized by bisected non-fucosylated oligosaccharides, promoting enhanced ADCC as a consequence of the affinity of FcγRIIIa as high as 50-fold or less (9). However, the carbohydrate modification introduced by overexpression of GnT III, which inhibits the fucosylation reaction, only sees only partial non-fucosylated antibodies. Since the Fc domain of an IgG molecule has two N-linked glycosylation sites, partial inhibition of the fucosylation reaction can lead to a variety of Fucose distributions within the antibody pool. Such antibody preparations will comprise one or two fucose residues, while some of which will comprise a mixture of fully non-fucosylated molecules. Clearly, this different degree of non-fucosylation affects the overall affinity for Fc [gamma] RIIIa, resulting in a different biological activity. Therefore, detailed characterization of the antibody pool is essential.

The affinity difference between fucosylated IgG and non-fucosylated IgG is less than 50-fold, so that the interaction can be used to separate different fucosylated species from the antibody pool and characterize them independently.

Conventional affinity chromatography substrates used for IgG purification were unable to distinguish different glycosylation patterns within the IgG pool because the immobilized capture protein specifically binds to the protein backbone of the antibody. For example, protein A and protein G bind at the junction between the CH2 and CH3 domains of the Fc region whereas other IgG specific proteins, such as protein L, recognize the constant portion of the kappa light chain (11-13).

Lecithin affinity chromatography using aleuria aurantiacetin (AAL), which binds fucose-containing glycans, for example, has been used to enrich proteins having a specific glycan structure (14) . Alternatively, glycan-target antibodies that recognize specific carbohydrate structures have been used, for example antibodies specific for the Lewis x antigen (15). These methods may be suitable to float the glycoprotein carrying a particular carbohydrate, but they are limited to be used in the flocculation of glycoproteins lacking certain carbohydrates such as non-fucosylated antibodies. Moreover, none of these methods is specific for the antibody, so strict purification of the antibody pool is required prior to application to an affinity substrate in order to avoid contamination by other proteins having a targeted glycan structure. Finally, these methods rely on specific lecithins or antibodies that may be difficult to obtain and have not been used successfully to meet manufacturing objectives.

Given its highly increased potential in inducing an immune effector function that is interesting for therapeutic purposes as well as therapeutic purposes, it is desirable to isolate a partial or complete bupeco-fucosylated antibody from a fully fucosylated antibody present in the antibody pool can do. The present invention provides a simple and efficient method for achieving such separation.

The technology of the present invention

The present invention provides a separation method based on the ability of a particular Fc receptor, such as Fc [gamma] RIIIa, to differentiate between a fucosylated antibody and a (partial or complete) non-fucosylated antibody. The method utilizes immobilized Fc receptors to separate different fucosylated antibodies from the antibody pool for analytical purposes as well as for manufacturing purposes. The methods described herein can be applied analytically to characterize the carbohydrate composition of the antibody pool. A large number of samples can be screened in this manner, for example, to select host cells producing a glycosylated antibody with a high content of non-fucosylated oligosaccharides. A manufacturing application allows the production of a complete bifocoxylated or fully fucosylated antibody population and can characterize its different Fc [gamma] RIIIa binding properties and biological activity.

In a first aspect, the present invention provides a method of separating an antibody having a different degree of fucosylation, comprising the steps of:

a) providing a population of antibodies,

b) contacting said antibody population with a fixed Fc receptor on a support,

c) eluting the antibody not specifically binding to said Fc receptor, and

d) eluting the antibody that specifically binds to said Fc receptor.

In certain embodiments, the antibody is an IgG antibody, more particularly an IgG ₁ antibody. In some embodiments, the antibody comprises a human Fc region. In one embodiment, the antibody is a glycosylated antibody. In certain embodiments, the antibody has an oligosaccharide engineered to alter its Fc region. In a more specific embodiment, the antibody is glycosylated to have an increased ratio of non-fucosylated oligosaccharides in its Fc region compared to the corresponding non-glycosylated antibody. In one embodiment, the antibody is administered in a host cell engineered to have increased? (1,4) -N-acetylglucosaminyltransferase (GnT) III activity as compared to non-engineered host cells It has been created. In a more particular embodiment, the host cell is further engineered to have an a-mannosidase II (ManII) activity. Increased GnTIII activity is typically induced by binding of one or more polypeptides having GnTIII activity to the host cell, i.e., N- linked < RTI ID = 0.0 > N- The introduction of one or more polynucleotides encoding polypeptides capable of catalyzing the addition of acetylglucosamine (GlcNAc). This is because, as determined in a specific biological assay, depending on the dose or not, the β-1,4-mannosyl-glycoprotein 4 (SEQ ID NO: (1,4) -N-acetylglucosaminyltransferase III, which is also known as beta-N-acetylglucosaminyl-transferase (EC 2.4.1.144) And a fusion polypeptide that exhibits an enzyme activity that does not occur. In certain embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the ooge localization domain of a heterologous Golgi resident polypeptide and the catalytic domain of GnTIII. In particular, the osteocalization domain is the localization domain of mannosidase II or GnTI, most particularly the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of: the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of the &agr; 1,6 central fucosyltransferase. A sugar manipulation method that can be used to manipulate an antibody to have a ratio of increased non-fucosylated oligosaccharide in its Fc region is described in U.S. Pat. 180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006), and PCT publications WO 99/54342, WO 2004/065540 and WO 03/011878.

In certain embodiments, the binding affinity of an Fc receptor for an antibody is dependent on the degree of fucosylation of the antibody. In certain such embodiments, the binding affinity of the Fc receptor for the antibody decreases with the degree of fucosylation of the antibody. In one embodiment, particularly where the antibody is an IgG antibody, the Fc receptor is an Fc? Receptor. In certain embodiments, the Fc receptor is Fc [gamma] RIIIa. In a more particular embodiment, the Fc receptor is Fc [gamma] RIIIa (V158). In some embodiments, particularly where the antibody comprises a human Fc region, the Fc receptor is human. In one embodiment, the Fc receptor is a recombinant Fc receptor (i. E., The Fc receptor is obtained by recombinant production).

In one embodiment, the support to which the Fc receptor is immobilized is a polymeric substrate. The polymer substrate is typically in the beaded form. In one embodiment, the polymer substrate is a crosslinked agarose or a derivative thereof. In certain embodiments, the polymer substrate is Sepharose (crosslinked agarose, available from GE Healthcare, Uppsala, Sweden). In another embodiment, the polymer substrate is a crosslinked poly (styrene-divinylbenzene). In certain embodiments, the polymer substrate is POROS® (crosslinked poly (styrene-divinylbenzene), available from Applied Biosystems, Foster City, USA). Fc receptors can be immobilized on a support in a variety of ways. Those skilled in the art will readily be able to determine suitable fixing methods based on the properties of the support. For example, immobilization on a polymer substrate is typically achieved through a chemical reaction of a protein immobilized on a functional group (e.g., a hydroxyl group, an aldehyde group, an epoxide group) contained in the substrate. Suitable polymeric substrates as well as coupling chemistry and protocols are well known in the art. Two different support phase immobilisations are also described in the following examples. In one embodiment, the support is not a cell or membrane. In certain embodiments, the method is a chromatographic method. In one such embodiment, a support is included in the chromatography column. In one such embodiment, contact of the antibody population with the Fc receptor is carried out by passing the antibody population through a chromatography column.

In certain embodiments, the population of antibodies is purified. In certain embodiments, the population of antibodies is affinity purified using affinity purification, particularly Protein A or Protein G. Affinity purification can be performed, for example, as a batch purification by incubation of affinity substrates and antibody populations, or as chromatographic purification by passing the antibody population mobile phase over an affinity substrate stationary phase. In one embodiment, the antibody population is purified by affinity chromatography. In a more specific embodiment, the antibody population is purified by protein A affinity chromatography, in particular protein A affinity chromatography. In other embodiments, the antibody population is purified by size exclusion chromatography, or by a combination of affinity chromatography and size exclusion chromatography.

In one embodiment, the population of antibodies is provided in solution. In a more specific embodiment, the population of antibodies is provided in a buffer solution. In one embodiment, the buffer solution has a pH value of from about 7.0 to about 8.5, especially a pH value of about 8. In one embodiment, the buffer solution is a Tris buffer solution. In a more particular embodiment, the buffer solution comprises 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 8, or 20 mM Tris, 20 mM MOPS (3- (N-morpholino) propanesulfonic acid) Citrate, 100 mM NaCl, pH 8. In some embodiments, contact of the antibody population with an Fc receptor is done in a buffer solution. In certain such embodiments, the buffer solution is identical to the buffer solution in which the antibody population is provided. In one embodiment, the antibody population is purified by protein A or protein G affinity chromatography, in particular protein A affinity chromatography, and contact with the Fc receptor allows the antibody population to be purified by affinity chromatography (from protein A to eluted antibody In the same buffer solution obtained after neutralization of the solution. In certain embodiments, an intermediate step between the purification of the antibody population and contact with the Fc receptor is not required.

In some embodiments, the method further comprises the steps of:

c1) Support cleaning.

In one embodiment, the cleansing comprises contacting the support with a buffer solution that maintains specific binding of the antibody to the Fc receptor, followed by removal of the buffer solution. In one embodiment, the buffer solution has a pH value of from about 7.0 to about 8.5, especially a pH value of about 8. In one embodiment, the buffer solution is a Tris buffer solution. In a more particular embodiment, the buffer solution comprises 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 8, or 20 mM Tris, 20 mM MOPS (3- (N-morpholino) propanesulfonic acid) Citrate, 100 mM NaCl, pH 8. In one embodiment, the buffer solution is the same as the buffer solution in which the antibody is provided. In one embodiment, the same buffer solution provides a (purified) population of the antibody therein (step a), contacting the population of the antibody with an Fc receptor (step b), an antibody that does not specifically bind to the receptor (Step c), and is used to clean the support (step c1).

In certain embodiments, the methods comprise administering an antibody subsp in which the majority of the antibodies do not comprise a fucose moiety at either of the two N-glycans of the antibody Fc region, or which contain a fucose moiety at one or both of them, - Allow separation of populations. In a further specific embodiment, the method comprises administering an antibody having a fucosylation degree of 90% or more, preferably 95% or more, an antibody having a fucosylation degree of less than 20%, preferably less than 10% %, Preferably about 20% to 60%, of the antibody. In one embodiment, the method is suitable for the separation of antibody sub-populations consisting essentially of partial fucosylation and fully non-fucosylated antibodies. In one embodiment, the method comprises administering an antibody sub-population consisting essentially of an antibody that does not comprise a fucose residue at either of the two N-glycans of the antibody Fc region, or that comprises a fucose residue at one of the two It is suitable for separation. In one embodiment, the method is suitable for the isolation of antibody sub-populations essentially free of antibodies comprising fucose residues in both of the two N-glycans of the antibody Fc region.

In certain embodiments, the method is for analytical purposes. In other embodiments, the method is for manufacturing purposes.

In certain embodiments, the method further comprises the steps of:

e) Collection of eluted antibodies of step c) and / or step d).

In some embodiments, the method further comprises the steps of:

f) using antibodies collected for experimental or therapeutic purposes.

In one embodiment, eluted antibodies are detected by measuring UV absorbance at a wavelength of 280 nm.

In one embodiment, elution of step c) comprises separation of the remaining antibody from the antibody population after contact in step b). When a support is included in the chromatography column and contact of the antibody population with the Fc receptor is carried out through the chromatography column of the antibody population, the eluted antibody in step c) is found in the passage liquid of the chromatography column . In one embodiment, the eluted antibody in step c) is a fully fucosylated antibody. In a more particular embodiment, the eluted antibody in step c) has a degree of fucosylation of 90% or more, preferably 95% or more. In one embodiment, the majority of the antibodies eluted in step c) comprise fucose residues in each of the two N-glycans of the antibody Fc region.

In certain embodiments, elution of step d) comprises contacting the support with a buffer solution that prevents binding of the antibody to the Fc receptor. In one such embodiment, the pH value of the buffer solution ranges from about 3 to about 5, preferably from about 4 to about 5. [ In one embodiment, the buffer solution is a Tris buffer solution. In one embodiment, the eluted antibody in step d) is a partially fucosylated and / or fully non-fucosylated antibody. In a more specific embodiment, the eluted antibody in step d) has a fucosylation degree of less than 90%, preferably less than 75%, most preferably less than 60%. In one embodiment, the majority of the eluted antibodies in step d) comprise fucose residues in one of the two N-glycans in the antibody Fc region, or none of them contain fucose residues.

In certain embodiments, elution of step d) is performed at different pH values. In one embodiment, the range of pH values is from about 3 to about 5, preferably from about 4 to about 5. In certain embodiments, the pH values include 4.6 and 4.2. In another specific embodiment, the elution of step d) allows separation of the partial fucosylated antibody and the fully non-fucosylated antibody. In a more particular embodiment, the elution of step d) is carried out in the presence of an antibody having a degree of fucosylation of less than 20%, preferably less than 10%, and an antibody having a degree of fucosylation of about 10% to 75%, preferably about 20% to 60% Allow separation. In yet another particular embodiment, the elution of step d) is performed such that the majority of the antibodies comprise fucose residues in one of the two N-glycans of the antibody Fc region, or the majority of the antibodies comprise two N - allows separation of antibody sub-populations not containing fucose residues anywhere in glycans. In some embodiments, elution of step d) is performed by continuously contacting the support with a series of buffer solutions that inhibit binding of the antibody with different degrees of fucosylation to the Fc receptor. In certain such embodiments, the buffer solution has different pH values. In one embodiment, the pH value ranges from about 3 to about 5, preferably from about 4 to about 5. In certain embodiments, the pH values include 4.6 and 4.2. In one embodiment, the buffer solution is a Tris buffer solution. In a more specific embodiment, the buffer solution comprises 10 mM Tris, 50 mM glycine, 100 mM NaCl, or 20 mM Tris, 20 mM MOPS (3- (N-morpholino) propanesulfonic acid), 20 mM sodium citrate, 100 mM NaCl (having a different pH value).

In a further aspect, the invention encompasses the use of an Fc receptor in a method of separating antibodies having different degrees of fucosylation. In one aspect, the invention provides the use of an Fc receptor in the methods of the invention described herein. In one embodiment, the Fc receptor is an Fcy receptor. In certain embodiments, the Fc receptor is Fc [gamma] RIIIa. In a more particular embodiment, the Fc receptor is Fc [gamma] RIIIa (V158). In one embodiment, the Fc receptor is human. In a further embodiment, the Fc receptor is a recombinant Fc receptor. In certain embodiments, the Fc receptor is immobilized on a support. The support may incorporate any of the features, alone or in combination, as described in the preceding paragraphs relating to the support used in the method of the present invention.

In another aspect, the invention provides a Fc receptor immobilized on a support for use in a method of separating antibodies having different degrees of fucosylation, such as the method of the present invention. In one embodiment, the Fc receptor is an Fcy receptor. In certain embodiments, the Fc receptor is Fc [gamma] RIIIa. In a more particular embodiment, the Fc receptor is Fc [gamma] RIIIa (V158). In one embodiment, the Fc receptor is human. In a further embodiment, the Fc receptor is a recombinant Fc receptor. The support may incorporate any of the features, alone or in combination, as described in the preceding paragraphs relating to the support used in the method of the present invention.

Unless otherwise defined, terms such as those commonly used in the art are used herein.

The term "antibody" is used in the broadest sense and refers to a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody) and an antibody fragment (Fc region of an immunoglobulin or Fc region of an immunoglobulin And the like), as well as various antibody structures. The term also encompasses fusion proteins comprising a region or Fc region equivalent to the Fc region of an immunoglobulin.

The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, the immunoglobulin of the IgG class is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two light chains and two heavy chains (disulfide-linked). (HH) and three constant domains (CH1, CH2, and CH3; also referred to as a variable heavy chain domain or heavy chain variable domain) in the N-terminus to the C- Also referred to as a heavy chain constant region). In the case of IgE class immunoglobulins, the heavy chain additionally has the CH4 domain. Thus, the immunoglobulin heavy chain is a polypeptide consisting of the following domains in the C-terminal direction from the N-terminus: VH-CHl-HR-CH2-CH3- (CH4). Likewise, in the C-terminal direction from the N-terminus, each light chain has a constant region (VL; also referred to as a variable light chain domain or light chain variable domain) followed by a constant light chain (CL) domain (also referred to as a light chain constant region) . Thus, the immunoglobulin light chain is a polypeptide consisting of the following domains in the C-terminal direction from the N-terminus: VL-CL. The immunoglobulin heavy chain is α (IgA), δ (IgD ), ε (IgE), γ (IgG), or μ (IgM) (some of which are sub-sub additional type: for example, γ ₁ (IgG ₁ ), it can be assigned to one of five types, called Zoned _{γ 2 (IgG 2), γ} 3 (IgG 3), γ 4 (IgG 4), α 1 (IgA 1) and α ₂ (IgA ₂₎₎ . The light chain of an immunoglobulin can be assigned to one of two types, referred to as kappa (kappa) and lambda (lambda), based on the amino acid sequence of the constant domain. Immunoglobulins consist essentially of two Fab fragments and an Fc region linked through an immunoglobulin overlap region.

"Class" of an antibody or immunoglobulin refers to a type of constant domain or constant region retained in its heavy chain. There are antibodies of the five major classes are: IgA, IgD, IgE, IgG , and IgM, some of which can be added divided into a sub-classes (isotypes) e.g., IgG _1, IgG _2, IgG _3, IgG _4, IgA _1, and IgA _2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as?,?,?,?, And?, Respectively.

The term "Fc region" or "Fc domain" is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes a native sequence Fc region and a variant Fc region. The boundaries of the Fc region of the IgG heavy chain may vary slightly, but it is customary that the human IgG heavy chain Fc region is usually defined to encompass Cys226 or Pro230 to the carboxyl-terminal end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. The so-called EU index, as described in the Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

By "region equivalent to the Fc region of an immunoglobulin" is meant a region that has altered substitution, addition or deletion as well as naturally occurring allelic variants of the Fc region of the immunoglobulin, but mediates effector function (e.g., antibody dependent cell mediated cytotoxicity) The ability of the immunoglobulin is intended to include variants that do not substantially decrease. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of the immunoglobulin without substantial loss of biological function. Such variants may be selected according to the general rules known in the art to have minimal effect on activity (see, e.g., Bowie et al., Science 247, 1306-10 (1990)).

The term "fucosylation" refers to the presence of fucose residues in the oligosaccharide attached to the peptide backbone of the antibody. Specifically, misfire fucose antibodies in the innermost part of the N- acetylglucosamine (GlcNAc) residues in both 1 or both of the N- linked oligosaccharides attached to antibody Fc regions, e.g., at a position Asn297 of human IgG ₁ Fc domain ( EU numbering of Fc region residues), alpha (1,6) -linked fucose. Asn297 may also be located about 29 amino acids up or down at position 297, i.e. between positions 294 and 300 due to small sequence changes in the immunoglobulin.

"Fucosylation degree" is the percentage of fucosylated oligosaccharide relative to all oligosaccharides identified in an N-glycosidase F treated antibody sample by MALDI TOF MS. In the sample of "complete fucosylating antibody ", essentially all oligosaccharides contain fucose residues, i.e. all oligosaccharides are fucosylated. In one embodiment, the fully fucosylated antibody has a fucosylation degree of 90% or greater. Thus, each antibody in such a sample typically contains a fucose residue in each of the two N-linked oligosaccharides in the Fc region. Conversely, in a sample of a "complete bifocoxylating antibody ", essentially none of the oligosaccharides is fucosylated, and each antibody in such a sample does not contain a fucose residue anywhere in the two N-linked oligosaccharides in the Fc region. In one embodiment, the fully non-fucosylated antibody has a degree of fucosylation of less than 10%. In the "partial fucosylated antibody" sample, only a portion of the oligosaccharide contains fucose. In this sample, each antibody may contain no fucose residues in any of the N-linked oligosaccharides in the Fc region, or may contain fucose residues in one or both of them, provided that essentially all of the sample Each antibody does not contain a fucose residue either in the N-linked oligosaccharide in the Fc region nor essentially every individual antibody in the sample contains a fucose residue in both of the N-linked oligosaccharides in the Fc region Points are conditional. In one embodiment, the partial fucosylated antibody has from about 10 to about 75% fucosylation.

As used herein, the term "glycosylated, sugar modified, glycosylated" refers to post-translational modification or any manipulation of a peptide backbone of a naturally occurring or recombinant polypeptide or fragment thereof that alters the glycosylation pattern of the polypeptide do. Amino acid sequences, side chain groups of each amino acid or oligosaccharide structure as well as combinations of these approaches. The glycocalyx also includes metabolic engineering manipulation of the glycosylated tissue of the cell, including achieving altered glycosylation of the glycoprotein expressed in the cell by genetic manipulation of the oligosaccharide synthesis pathway. In addition, it includes mutations to the glycosylation of the sugar chains and the action of the cellular environment. In one embodiment, the engineered antibody is directed against altering glycosyltransferase activity in the host cell producing the antibody. Examples of glycosyltransferases include β (1,4) -N-acetylglucosaminyltransferase III (GnTIII), β (1,4) -galactosyltransferase (GalT), β , 2) -N-acetylglucosaminyltransferase I (GnTI),? (1,2) -N-acetylglucosaminyltransferase II (GnTII) and? (1,6) -fucosyltransferase Aze. In certain embodiments, the engineered antibody is directed against altered glucosaminyl transferase activity and / or fucosyltransferase activity in the host cell producing the antibody. Antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may be used, for example, with an increased [beta] (l, 4) -N-acetylglucosaminyl transferase III (GnTIII) Can be obtained by generating antibodies in host cells with mannosidase II (ManII) activity or in host cells with reduced [alpha] (1,6) fucosyltransferase activity.

"FcγRIIIa (V158)" refers to the subtype of FcγRIIIa (also known as CD16a; Uni Prot No. P08637, NCBI Accession No. NP_000560, human protein) with a valine (V) residue at amino acid position 158. IgG binding by FcγRIIIa (V158) was found to be superior to binding by FcγRIIIa (F158) (17).

By "specifically bound" is meant that the binding can be distinguished from selective and unwanted or non-specific interactions with Fc receptors. The ability of an antibody to bind to an Fc receptor can be measured by enzyme immunoassay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) as described herein.

"Binding affinity" refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between binding pair members (eg, a receptor and its ligand). The affinity of the molecule X for its partner Y can generally be expressed by the dissociation constant (K _D ), which is the ratio of dissociation and association rate constants (k _off and k _on , respectively). Thus, the equivalent affinity may include a different rate constant as long as the ratio of the rate constants remains the same. The affinity may be measured by well-established methods known in the art, including those described herein. Specific methods for affinity measurement include surface plasmon resonance (SPR).

When used in connection with an antibody population, the term "purified" means that the antibody population is not necessarily associated with an unrelated non-antibody protein. Various methods for the purification of antibodies are known in the art, including high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. An "affinity purified" antibody population is defined as an affinity matrix in which antibodies, but not unrelated non-antibody proteins, specifically bind to an affinity substrate such as protein A or protein G, ≪ / RTI >

As used herein, the term "buffer solution" refers to a solution having a specified pH value, typically a buffering agent to stabilize the pH of the solution. Buffers are well known in the art and include, for example, citrate salts, acetate salts, histidine salts, succinate salts, maleate salts, phosphate salts, lactate salts or tris (hydroxymethyl) amino- ).

As used herein, "majority" means more than 50%, preferably more than 60%, most preferably more than 70%, of the total.

Figure 1. N-linked oligosaccharide attached to Asn297 of the human IgGl Fc domain. The bivalent sugars define the center of the pentasaccharide saccharide, and the additives of other saccharide residues are variable. GlcNAc: N-acetylglucosamine; Fuc: fucose; Man: Mannose; Gal: Galactose; NeuAc: N-acetylneuraminic acid.
Figure 2. Purification of soluble human Fc [gamma] RIIIa (V158). A) Chromatogram of immobilized metal chelate chromatography (IMAC). Solid line: A _{280 nm} ; Dotted line: Gradient. B) Chromatogram of size exclusion chromatography (SEC). Solid line: A _280nm . C) SDS PAGE, Kusami staining. Lane 1: molecular weight marker [kDa]; Lane 2: Reduced FcγRIIIa (V158). D) Analytical SEC chromatogram (A _{280 nm} ). 50 샘플 samples were injected.
Figure 3. Analytical FcγRIIIa (V158) chromatography. 10 [mu] g A) engineered IgG "A" B) wild-type IgG "A "; C) engineered IgG "B" D) Chromatogram for wild type IgG "B " (A _{280 nm} ). Peaks corresponding to antibody fractions with a high content of bifunctional glycans ("binding peaks") are indicated by black squares. The area of the "binding peak" is as follows: A) 66% of the total peak area (48% bifunctional as measured by MALDI TOF MS); B) 26% of the total peak area (as determined by MALDI TOF MS, 10% bifunctional); C) 75% of the total peak area (75% bifunctional as measured by MALDI TOF MS); D) 31% of the total peak area (9% bifunctional as measured by MALDI TOF MS).
Figure 4. Evaluation of analytical FcγRIIIa (V158) chromatography. Percentage of area of the "binding peak" from the chromatographic column of FcγRIIIa (V158) of the total peak area as a function of the percentage of bifunctional as measured by MALDI TOF MS. A) sugar manipulation and a mixture of wild type IgG "A" (0-100% engineered IgG). B) per gram and a mixture of wild type IgG "B" (0-100% engineered IgG).
Figure 5. FcγRIIIa (V158) after MALDI TOF MS and protein A chromatography after protein A chromatography as two different methods to analyze the degree of fucosylation of the purified antibody (sugar engineered IgG "C") from the cell culture supernatant. Comparison of chromatography. Percentage of the area of the "binding peak" from the FcγRIIIa (V158) chromatography column of the total peak area as a function of the percentage of non-fucosylated as measured by MALDI TOF MS.
6. FcγRIIIa (V158) chromatography for preparation. Chromatogram of the engineered IgG "A ". IgG "A" elutes at 3 peaks: Peak 1 elutes to the column through the column, Peak 2 and 3 to two pH steps. 0.0 > 1, < / RTI > 2 and 3, respectively. Solid line: A _{280 nm} ; Dotted line: gradient; Dash line: pH-value.
Figure 7. Biological activity of antibody fractions collected from preparative FcγRIIIa (V158) chromatography. ADCC assays were performed on the three eluted peaks as well as the starting material for the engineered IgG "A" (A) and IgG "B" (B) (antibody pool as loaded on FcγRIIIa (V158) Respectively. Black square: starting material; White Diamonds: Peak 1; Black Triangle: Peak 2; Black circles: Peak 3; White squares (only for B): Wild type IgG "B" (not per-manipulated).
Figure 8. Interaction of soluble human Fc [gamma] RIIIa (V158) with antibody fractions collected from preparative Fc [gamma] RIIIa (V158) chromatography, analyzed by surface plasmon resonance. A) IgG "A ", Peak 1 (analyzed in steady state); B) IgG "B ", peak 2; C) IgG "B ", peak 3; D) Sensorgrams and fittings for antibody pools of IgG "B" as loaded on FcγRIIIa (V158) columns. E) Sensorgram of wild-type IgG "B " without peritoneal (analyzed in steady state).

Example

The following is a method embodiment of the present invention. It will be apparent that various other embodiments may be practiced in light of the general description provided above.

Example 1

Soluble human Fc [gamma] RIIIa (V158) K ₆ H ₆  ≪ / RTI >

Soluble human FcγRIIIa (V158) with C-terminal (lysine) ₆ and (histidine) ₆ tags (see SEQ ID NOs 1 and 2) was transfected with a mammalian expression vector using calcium phosphate transfection to transfect HEK293-EBNA cells Lt; / RTI >

For transfection, cells were grown as adhered monolayer cultures in a T-flask using DMEM culture medium supplemented with 10% (v / v) FCS and the cells were seeded at 50-80% confluent Lt; / RTI > In the transfection of T150 flasks, 15 million cells were seeded 24 h prior to transfection in 25 ml DMEM culture medium supplemented with FCS (final 10% v / v) and the cells were seeded in a humidified 5% CO ₂ atmosphere , Placed in a 37 < 0 > C incubator overnight. For each T150 flask to be transfected, a solution of DNA, CaCl _2, and water was prepared by mixing 94 μg total plasmid vector DNA and 469 μl 1 M CaCl ₂ solution in water to a final volume of 469 μl. To this solution, add 938 μl of 50 mM HEPES, 280 mM NaCl, 1.5 mM Na ₂ HPO ₄ solution, pH 7.05, mix immediately for 10 seconds, and then stand at room temperature for 20 seconds. The suspension was diluted with 10 ml of DMEM supplemented with 2% (v / v) FCS and added to the T150 flask instead of the original medium. Then, an additional 13 ml of transfection medium was added. Of the cells 37 ℃, in 5% CO ₂ and incubated for about 17 to 20 hours, the medium was replaced with 25 ml DMEM, 10% FCS. Conditioned culture medium was collected after centrifugation at 210 xg for about 7 days after medium-exchange, and the solution was sterile filtered (0.22 占 퐉 filter) and sodium azide was added at a final concentration of 0.01% (w / v) And kept at 4 [deg.] C.

The secreted proteins were purified by immobilized metal chelate chromatography (IMAC) and size exclusion chromatography (Fig. 2A, B).

For metal chelate chromatography, the supernatant was loaded at 4 ml / min on a NiNTA Superflow cartridge (column volume 5 ml; Qiagen, Germany) equilibrated with buffer A (20 mM Na ₂ HPO ₄ 0.5 M NaCl pH 7.4) . Unbound protein was removed by washing with at least 10 column volumes Buffer A. FcγRIIIa (V158) was eluted with a gradient to buffer B (20 mM Na ₂ HPO ₄ , 0.5 M NaCl, 0.5 M imidazole, pH 7.4). The gradient consists of the following three steps: 1) 0 to 45% Buffer B on 8 column volumes, 2) 45 to 100% Buffer B on 2 column volumes, and 3) 100% buffer on 2 columns. B. Second elution peak Was pooled and loaded onto a size exclusion chromatography column (HiLoad 16/60 Superdex 75 (GE Healthcare, Sweden) equilibrated with 2 mM MOPS, 150 mM NaCl, 0.02% (w / v) NaN ₃ , And concentrated using a centrifugal filter unit (Amicon Ultra MWCO 10 kD; Millipore, USA).

The protein concentration of the purified protein sample was determined by measuring the absorbance (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of the recombinant human Fc [gamma] RIIIa (V158) was analyzed by SDS PAGE and InstantBlue ™ (Expedeon) staining in the presence and absence of reducing agent (5 mM 1,4-dithiothreitol). The NuPAGE® Pre-Cast Gel System (4-12% Bis-Tris, Invitrogen, USA) was used according to the manufacturer's instructions (FIG. 2C). Protein aggregate content (50 ug samples) was analyzed using a Superdex 75 10 / 300GL analytical size exclusion column (GE Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w / v) NaN ₃ , , ≪ / RTI > at 25 C (Figure 2D).

Example 2

Analysis of antibody fucosylation

Preparation of Fc fragments from human IgG. The antibody was incubated at 25 DEG C for 72 hours in 50 mM Tris pH 8.0, 150 mM NaCl (Roche, Switzerland) containing 0.42 U of plasmin per mg. The cleaved Fc fragments were separated from Fab-fragments using protein A beads (GE Healthcare) washed with 50 mM Tris pH 8.0, 100 mM glycine, 150 mM NaCl. The Fc fragment was eluted with 50 mM Tris pH 3.0, 100 mM glycine, 150 mM NaCl. The eluant was neutralized by addition of 1:40 v / v 2 M Tris pH 8.0 and loaded onto a size exclusion chromatography column (Superdex S200 10/300 GL, GE Healthcare). The sample was concentrated and the buffer was exchanged with 20 mM Tris pH 8 (Amicon, Millipore).

Release of N-Linked Oligosaccharides from Human Fc Fragments Different enzymes were used to hydrolyze N-linked glycans from human Fc fragments. N-linked oligosaccharide was cleaved from 1 mg of Fc fragment by incubation with 0.005 U recombinant PNGase F (QAbio, USA). To release carbohydrates from Fc fragments using non-tagged Endo S (Genovis, Sweden) and Endo H (QAbio, USA), samples were incubated with Endo S and 0.1 U / mg combined at a 1:20 molar ratio Incubated with Endo H. All reactions were incubated for 16 hours at 37 < 0 > C in 20 mM Tris pH 8.0.

Release of N-linked oligosaccharides from whole human IgG. Samples were buffer exchanged in 20 mM Tris pH 8.0 (Amicon 5.000 MWCO, Millipore) in order to release carbohydrates from whole human IgG using untagged Endo S and Endo H, which were incubated with Endo S and 0.1 U / mg Endo H at 37 < 0 > C for 16 hours in a 1: 7 molar ratio combination.

The samples were further incubated with carboxypeptidase B (Roche; 1 mg / ml) to eliminate heterogeneity caused by carboxypeptidase B-treated C-terminal lysine. Thus, 1 [mu] l of carboxypeptidase B per 50 [mu] g protein was added to the endoglycosidase reaction and incubated again at 37 [deg.] C for 1 hour. After digestion, the sample was purified using Protein A (POROS A 20, Applied Biosystems) and neutralized with 1:40 v / v 2 M Tris pH 8.0.

MALDI-TOF mass spectrometry. Neutral oligosaccharide profiles for human IgG were analyzed by mass spectrometry (Autoflex, Bruker Daltonics GmbH) in positive ion mode according to Papac et al (18).

ESI-MS analysis of glycan structure from human Fc fragments by direct injection (off-line detection). Treated with protease plasmin and carboxypeptidase B and with endoglycosidase Endo S and Endo H 20-50 [mu] g (no more than 90 [mu] l) of the antibody was applied to a Sephadex G25 self-packaging ECO SR column (5 x 250 mm) (KronLab®) equilibrated with 2% (v / v) formic acid, 40% (v / v) acetonitrile ) At a flow rate of 0.5 ml / min for 30 minutes. The injected antibody samples were desalted using an 8 minute isocratic elution with 2% (v / v) formic acid, 40% (v / v) acetonitrile at a flow rate of 1 ml / min. Elution of desalted protein was recorded at 280 nm with UV and elution samples (about 200-300 [mu] l volume) were collected in a 1.5 ml reaction vial. The desalted sample aliquot was passively filled into a metal-coated glass needle (Proxeon Biosystems Nano ESI-needles, cat # ES387), inserted into the nano spray source of the mass spectrometer and analyzed using Waters ESI-Q-TOF II mass spectrometry Or to a Q-Star Elite mass spectrometer from Applied Biosystems. MS spectra were obtained using a capillary voltage of 1000 V and a cone voltage of 30 V in the range of 1000-2000 m / z mass in positive ion mode using a source temperature of 80 캜. The desolvation temperature did not work. MS data was acquired by each device software for 2-3 minutes. Dimeric Fc fragments (i.e., molecules in which both peptide chains contain only N-acetylglucosamine residues (GlcNAc / GlcNAc)), a peptide chain comprising a different combination of glycan structures truncated by endoglycosidase application (GlcNAc + Fuc / GlcNAc) having a fucose residue and a molecule (GlcNAc + Fuc / GlcNAc + Fuc) in which both peptide chains have fucose residues) was further amplified using in house development software Fc < / RTI > fragment species. The relative contrast of the three different surrogate glycosylated dimeric Fc-fragments was calculated by the same in-house software using the sum of the peak areas of the m / z spectra of the different glycosylation variants.

ESI-MS analysis of the glycan structure from human Fc fragments by LC / MS (on-line detection). The LC-MS method was performed on an Agilent Cap LC1100 (Waters) connected to a QTof II mass spectrometer. Chromatographic separation was performed on a Phenomenex Jupiter C18 column (5 탆 particle size, 300 A pore size, 1 x 25 mm). Eluent A is 0.5% (v / v) formic acid in water and Eluent B is 70% (v / v) isopropanol, 20% (v / v) acetonitrile, 9.5% v) formic acid. The flow rate was 40 [mu] l / min and separation was carried out at 75 [deg.] C with 2 [mu] g protein in a final volume of 10 [mu] l.

Example 3

Analytical FcγRIIIa chromatography

Preparation of affinity substrates. 10 mg of FcγRIIIa (V158) was buffer exchanged with 0.1 M sodium phosphate, 0.05% (w / v) NaN ₃ , pH 7 using a centrifugal filter device (Amicon Ultra MWCO 10 kD; Millipore, USA) ml. < / RTI > The protein concentration was determined with a UV spectrometer measuring the absorbance at 280 nm and adjusted to 8 mg / ml. 0.14 g of dry beads and the corresponding 440 [mu] l of POROS AL beads (Applied Biosystems, USA) were added to the protein solution. Subsequently, 0.01 M was added to 1 M NaCNBH ₃ 41.5 ㎕ of NaOH, and incubating the suspension overnight at room temperature. The supernatant was removed by centrifugation of the beads, and the non-bound protein was quantitated with a UV spectrometer. The beads were quenched at room temperature for 30 minutes with 500 μl 1 M Tris, pH 7.4 and 23 μl 1 M NaCNBH ₃ (in 0.01 M NaOH). The beads were washed four times with 1 M NaCl and washed three times with 2 mM MOPS, 150 mM NaCl, 0.02% (w / v) NaN ₃ , pH 7.3. Finally, 14 mg of Fc [gamma] RIIIa (V158) was coupled per gram of POROS AL beads.

Analytical chromatography using POROS AL phase fixed FcγRIIIa (V158). POROS AL beads with Fc [gamma] RIIIa (V158) were packaged in a 2x20 mm Upchurch Scientific column (column volume: 60 [mu] l) mounted on an Agilent 1200 HPLC system (Agilent Technologies, USA). The buffer used was 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 8 for equilibration and washing, or 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 3 for elution. The pump flow rate of the system was set at 0.5 ml / min. At 0 hour, the antibody preparation (10 ug of protein A-purified antibody) was injected with an autosampler and rinsed for 2 minutes and then eluted with a step gradient of 0.66 minute period before re-equilibration for 4.33 minutes. The total cycle duration was 7 minutes.

The chromatogram showed two peaks: the passage peak and elution peak (Figure 3). The area of both peaks was measured as integral and the percentage of the eluted peak ("binding peak") relative to the total area was recorded.

To apply the method for high throughput analysis purposes and to quantify the percentage of non-fucosylation in the antibody preparation, the sample was first purified from the supernatant by affinity chromatography using Protein A on an Agilent 1200 HPLC system, Collected from the plate. Samples were eluted in 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 3, neutralized by addition of 1:40 (v / v) 2 M Tris pH 8 and re-injected onto FcγRIIIa (V158) Respectively. Since the antibody concentration is known after protein A chromatography, the injection volume was adjusted to inject 10 [mu] g of each sample on an Fc [gamma] RIIIa (V158) chromatography column.

The wild-type and glycosylated antibodies were mixed in the washing buffer to obtain different non-fucosylation ratios and chromatographically analyzed directly on a FcγRIIIa (V158) column. The percentage of non-fucosylated as measured by MALDI TOF MS and the percentage of "binding peak" on FcγRIIIa (V158) chromatography column shows a linear correlation (FIG.

 Cell culture supernatants of different cell clones expressing the engineered IgG "C " were simultaneously analyzed in two different ways: 1) MALDI TOF MS after protein A chromatography and 2) protein A chromatography followed by FcγRIIIa (V158) chromatography. For this high-throughput analysis, 100 [mu] l of supernatant was injected onto the protein A chromatography column, the eluant was neutralized by the addition of 1:40 (v / v) 2 M Tris pH 8 and the MALDI TOF MS analysis of the carbohydrate , Or injected onto FcγRIIIa (V158) chromatography columns. An advantage of the combination of protein A chromatography and FcγRIIIa (V158) chromatography is that the 96-well plate containing the eluted sample from the protein A column can be used directly after neutralization without any additional buffer exchange or pipetting steps . The percent area of the "binding peak" on the FcγRIIIa (V158) column was compared to the percentage of non-fucosylation obtained by MALDI TOF MS for antibodies produced by different cell clones. Similar ranking was obtained in both ways and it was shown that the method of the present invention enabled the identification of clones producing antibodies with the highest degree of non-fucosylation (Fig. 5).

These results show that Fc [gamma] RIIa chromatography can be used to screen cell culture supernatants at high throughputs and can rank the antibodies generated according to the degree of non-fucosylation.

Example 4

FcγRIIIa chromatography for preparation

Preparation of affinity substrates. 30 mg Fc [gamma] RIIIa (V158) was coupled to NHS activated Sepharose 4FF (GE Healthcare, Sweden). Briefly, FcγRIIIa (V158) was exchanged with 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.2, concentrated to a final volume of 2 ml, and washed with 3 ml NHS And incubated with activated beads. The supernatant was removed and the beads were incubated for an additional 2 hours at room temperature with 0.1 M Tris, pH 8.5. The beads were then packaged in an empty Tricorn 5/150 column (GE Healthcare, Sweden) as gravity flow and then packaged at 1.2 ml / min using Aekta Explorer 10 (GE Healthcare, Sweden). The final column volume was 2.7 ml at a column length of 14 cm. 30 mg human Fc [gamma] RIIIa (V158) was fixed.

Separation for the preparation of antibodies with different degrees of non fucosylation using FcγRIIIa (V158) immobilized on NHS Sepharose 4 FF. For chromatography, the column was equilibrated with 10 column volumes of 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, pH 8, and 3 mg of purified antibody (protein A affinity chromatography and size exclusion Chromatography) was loaded at a flow rate of 0.1 ml / min. The column was washed for 5 column volumes with 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, pH 8 and the different antibody populations were eluted with three pH steps of pH 4.6, pH 4.2 and pH 3 (Fig. 6). The desired pH value was determined by adding 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, pH 8 and 20 mM Tris, 20 mM MOPS, 20 mM sodium citrate, 100 mM NaCl, Mixed. The peaks were collected and concentrated and either injected onto protein A HPLC for purification, or batch purified with protein A beads (requiring subsequent MALDI TOF MS analysis). The antibody was subsequently analyzed for its carbohydrate composition, its binding to FcγRIIIa (V158), and its ability to induce ADCC.

Example 5

Analysis of isolated antibodies

Carbohydrate composition analysis. For MALDI TOF MS analysis of oligosaccharides, oligosaccharides were cleaved from antibodies purified with PNGase F and Endo H (16). FcγRIIIa (V158) chromatography, fractions of antibodies with different amounts of non-fucosylated glycans were isolated. The first peak corresponding to the passage liquid had the least amount of bifucosylated oligosaccharides, followed by peaks 2 and 3 (see Tables 2 and 3). However, MALDI TOF MS analysis shows only the total amount of non-fucosylated oligosaccharides in the formulation.

To determine the distribution of the two antibody heavy chain fucose residues in the Fc domain, the samples were digested with a combination treatment with Endo S and Endo H (described above and in PCT Publication No. WO 2011/039150, Which is incorporated by reference in its entirety).

In IgG "A ", the purified antibody was digested with plasmin, Endo H and Endo S to obtain a first N-acetylglucosamine residue centered on the oligosaccharide and an Fc fragment bearing fucose residues in the case of fucosylated carbohydrates did. These Fc fragments were analyzed by ESI-MS and the distribution of fucose per Fc fragment was determined (Table 1).

Table 1. Content of non-fucosylated carbohydrates for antibody pools of IgG "A" isolated in three fractions by FcγRIIIa (V158) chromatography. The degree of bupuchosylation was measured comprehensively by the MALDI TOF MS (mean of 7 runs) after PNGase F treatment, or the Fucosylation per Fc was measured by ESI-MS after the digestion with Plasmin / Endo S / Endo H (A pool of three times).

* Percentage of fucose residue-free glycans among all glycans eluted at each peak.

⁺ Calculated value for comparison of MALDI TOF and ESI-MS results. This value is calculated by adding the percentages of fucose-deficient glycans in all three Fc glycoforms. For example, at peak 2, the Fc fragment of 22/100 contains two non-fucosylated glycans (i.e., 44/200 glycans are non-fucosylated) and the 64/100 Fc fragment contains one non- (Ie, 64/200 glycans are non-fucosylated) and 14/100 Fc fragments do not contain any non-fucosylated glycans (ie, 0/200 glycans are bi fucosylated ), A total of 44 + 64 + 0 = 108/200 = 54%, non-fucosylated glycan eluted at peak 2.

In IgG "B ", the purified antibody was digested with Endo H (QA Bio) and Endo S (Genovis) to obtain a glycan composed of a first N-acetylglycosamine residue centered on oligosaccharides with or without fucose RTI ID = 0.0 > IgG. ≪ / RTI > These samples were analyzed by ESI-MS and the distribution of fucose per antibody was determined (Table 2).

Table 2. The content of non-fucosylated carbohydrates for antibody pools of IgG "B " isolated by three fractions by FcγRIIIa (V158) chromatography. The degree of non-fucosylation was measured comprehensively (by means of two rounds) after PNGase F treatment or by MALDI TOF MS, or by the ESI-MS after Endo S / Endo H digestion of the Fucose distribution per IgG Average).

* Percentage of fucose residue-free glycans among all glycans eluted at each peak.

⁺ Calculated value for comparison of MALDI TOF and ESI-MS results. This value is calculated by adding the percentages of fucose-deficient glycans in all three Fc glycans. For example, at Peak 2, the Fc fragment of 20.5 / 100 contains two non-fucosylated glycans (i.e., 41/200 glycans are non-fucosylated) and the 68.5 / 100 Fc fragment contains one non- (Ie, 68.5 / 200 glycans are bifucosylated) and the 11/100 Fc fragment does not contain any non-fucosylated glycans (ie, 0/200 glycans are bifucosylated ), Total 41 + 68.5 + 0 = 109.5 / 200 = 54.8%, non-fucosylated glycan eluted at peak 2.

For both IgG "A" and "B ", peak 1 contains most of the antibody (i.e., a fully fucosylated antibody) with a fucosylated sugar on both heavy chains in the Fc domain, while peak 2 contains one Fucosylation and one non-fucosylated carbohydrate, and the population of peak 3 comprises the majority of the complete non-fucosylated antibodies. Since the calculated values (see the last row in Tables 1 and 2 above) are generally less than the MALDI TOF MS results, the percentage of fully non-fucosylated antibodies appears to be slightly underestimated by the ESI-MS method.

Antibody-dependent cell-mediated cytotoxicity (ADCC). Different fractions were tested for their ADCC inducing ability. Raji (IgG "A" ADCC assay) or A549 (IgG "B" ADCC assay) cells were harvested (adherent cells with trypsin / EDTA) and washed and labeled with Calgene (Invitrogen) for 30 min at 37 ° C. After 30 minutes, the cells were washed three times with AIM V medium and resuspended in AIM V medium. They were plated at a concentration of 30,000 cells / well in a 96-well round-bottomed plate. Each antibody dilution was added and incubated for 10 minutes before contact with human effector cells (NK92 1708 clone LC3 E11, NK92 cells transfected with FcγRIIIa (V158)). The effector and target cells were co-incubated at 37C for 4 hours at a 3: 1 ratio. Lactate dehydrogenase (LDH) release was measured using the LDH cytotoxicity detection kit (Roche Applied Science, cat. No. 11 644 793 001). Calcine retention was confirmed by cleavage of the remaining cells with borate buffer (5 mM borate, 0.1% (v / v) Triton X-100) followed by Calcene fluorescence measurement. For the generation of antibody-dependent killing, spontaneous release (only antibody-free target and effector cells) was set to 0% kill and the maximal release (target cells and 2% (v / v) Triton X- It was set to death.

Only the first peak (with antibodies with both heavy chain fucosylated carbohydrates) had a reduced ADCC-inducing ability comparable to wild type IgG (Figure 7). Both peaks 2 and 3 have similar ADCC inducing ability, indicating that only one non-fucosylated glycan per antibody is sufficient to confer an excellent ADCC capability on IgG.

FcγRIIIa binding by surface plasmon resonance. Surface plasmon resonance was measured at 25 캜. Human antigen "B" was immobilized by amine coupling on a CM5 chip according to the manufacturer's instructions (GE Healthcare, Sweden). The IgG fraction was captured at 100 nM for 10 seconds at 10 ㎕ / min for 90 seconds. Human Fc [gamma] RIIIa (V158) was passed through the flow cells for 120 seconds at a flow rate of 50 l / min in the concentration range of 1.95-500 nM. The dissociation was monitored for 200 seconds. The surface was regenerated with two 10 mM glycine, pH 2 injections for 60 seconds before the next injection. The bulk refractive index difference was corrected by subtracting the response obtained on the reference flow cell. The association rate (k _on ) and dissociation rate (k _off ) were determined using a simple one-to-one Langmuir coupling model (BIACORE® T100 Evaluation Software version 1.1.1) with RI = 0 and Rmax = At the same time. The equilibrium dissociation constant (K _D ) is calculated as the ratio k _off / k _on .

The results obtained by surface plasmon resonance were consistent with the results of the ADCC assay (Table 3 and Figure 8). The wild-type IgG " B "as well as the first peak have a K _D of about 50 nM with very rapid on and off rates, whereas the peptides 2 and 3 and the sugar modified IgG" B " ) With a K _D of about 3 nM, the dissociation rate was much slower. Peak 3 IgG had the highest affinity for FcγRIIIa (V158).

Table 3. Affinity between FcγRIIIa (V158) and IgG "B". K _D obtained by surface plasmon resonance at < RTI ID = 0.0 > 25 C. < / RTI > Three pooled IgG "B" antibody pools separated by starting material and FcγRIIIa (V158) chromatography were captured on the immobilized antigen and FcγRIIIa (V158) was used as the degradation product. Fit: kinetic (1: 1 coupling RI = 0, Rmax = local) or steady state.

Taken together, these examples indicate that FcγRIIIa (V158) chromatography makes the antibody separable depending on the content of non-fucosylated carbohydrates in the Fc domain. The methods include screening the supernatant with a high-throughput (in combination with protein A chromatography), identifying clones producing antibodies with high degrees of non-fucosylation, or fractionating IgG according to fucose content for further characterization And can be applied to separation. Analysis of antibody populations separated by FcγRIIIa (V158) chromatography showed that IgG with most 1 or 2 bifocoxylated carbohydrates in the Fc domain behaves identically in terms of ADCC, so that 100% For the first time, that a population of bupuocyric antibodies is not required.

references

1. Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15 (1): 26-32

2. Krapp S et al. (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol. 325 (5): 979-89

3. Raju TS (2008) Terminal sugars of Fc glycans and the effector functions of IgGs. Curr Opin Immunol. 20 (4): 471-8

4. Jefferis R (2009) Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol Sci. 30 (7): 356-62

5. Hodoniczky J, Zheng YZ, and James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog. 21 (6): 1644-52

6. Malhotra R et al. (1995) Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement to the mannose-binding protein. Nat Med. 3: 237-43

7. Shields RL et al. (2002) Lack of fucose on human IgG ₁ N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J Biol Chem. 277 (30): 26733-40

8. Umana P et al (1999) Engineered glycoforms of an antineuroblastoma IgG ₁ with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol. 17 (2): 176-80

9. Ferrara C et al. (2006) Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous β (1, 4) -N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Biotechnol Bioeng. 93 (5): 851-61

10. Kanda Y et al. (2006) Comparison of biological activity among non-fucosylated therapeutic IgG ₁ antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology. 17 (1): 104-18

11. Deisenhofer J et al (1978) Crystallization, crystal structure analysis and atomic model of the complex formed by a human Fc fragment and fragment B protein A from Staphylococcus aureus. Hoppe Seylers Z Physiol Chem. 359 (8): 975-85

12. Groneborn AM and Clore GM (1993) Identification of the contact surface of a streptococcal protein G domain complexed with a human Fc fragment. J Mol Biol. 233 (3): 331-5

13. Graille M et al. (2001) Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural binding proteins of Fab binding proteins. Structure. 9 (8): 679-87

14. Drake P et al (2011) A lectin affinity workflow targeting glycoside-specific, cancer-related carbohydrate structures in trypsin-digested human plasma. Anal Biochem. 408: 71-85.

15. Cho W et al. (2008) Use of glycan targeting antibodies to identify cancer-associated glycoproteins in breast cancer patients. Anal Chem. 80: 5286-5292.

16. Schuster M et al (2005) Improved effector functions of a therapeutic monoclonal Lewis Y-specific antibody by glycoform engineering. Cancer Res. 65 (17): 7934-41.

17. Koene HR et al (1997) FcγRIIIa-158V / F polymorphism influences the binding of IgG by natural killer cell FcγRIIIa, independently of the FcγRIIIa-48L / R / H phenotype. Blood. 90: 1109-1114.

18. Papac DI et al. (1996) Analysis of acidic oligosaccharides and glycopeptides by matrix assisted laser desorption / ionization time-of-flight mass spectrometry. Anal Chem. 68: 3215-3223.

* * *

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is to be understood that the invention is not to be interpreted as limiting the scope thereof. The disclosures of all patents and scientific references cited herein are expressly incorporated by reference in their entireties.

                         SEQUENCE LISTING <110> Roche Glycart AG   <120> Separation method for fucosylated antibodies <130> 30672 &Lt; 150 > EP 11185798.3 <151> 2011-10-19 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 222 <212> PRT <213> Artificial Sequence <220> Soluble FcgRIIIa (V158) - (Lys) 6- (His) 6 fusion protein <400> 1 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro             20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln         35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu     50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln         195 200 205 Gly Lys Lys Lys Lys Lys Lys Lys Gly His His His His His     210 215 220 <210> 2 <211> 669 <212> DNA <213> Artificial Sequence <220> Soluble FcgRIIIa (V158) - (Lys) 6- (His) 6 fusion protein, DNA <400> 2 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaaggcaaa aagaaaaaga aaaagggcca ccaccatcac 660 catcactga 669

Claims (26)

A method for separating antibodies having different degrees of fucosylation comprising the steps of:
a) providing a population of antibodies,
b) contacting the population of said antibody with a fixed Fc receptor on a support,
c) eluting an antibody that does not specifically bind to said Fc receptor, and
d) eluting an antibody that specifically binds to said Fc receptor. 2. The method of claim 1 wherein the binding affinity of the Fc receptor for the antibody is dependent on the degree of fucosylation of the antibody. 3. The method according to claim 1 or 2, wherein the Fc receptor is an Fc? Receptor. 4. The method according to any one of claims 1 to 3, wherein the Fc receptor is Fc [gamma] RIIIa. 5. The method according to any one of claims 1 to 4, wherein the Fc receptor is Fc [gamma] RIIIa (V158). 6. The method according to any one of claims 1 to 5, wherein the antibody is an IgG antibody. 7. The method according to any one of claims 1 to 6, wherein the antibody is sugar-manipulated to have an increased ratio of non-fucosylated oligosaccharides in its Fc region as compared to the corresponding non-glycosylated antibody . 8. The method according to any one of claims 1 to 7, wherein the population of antibodies is purified. 9. The method according to any one of claims 1 to 8, wherein the population of antibodies is affinity purified using protein A or protein G. 10. The method according to any one of claims 1 to 9, wherein the support is a polymer substrate. 11. The method according to any one of claims 1 to 10, wherein the support is included in the chromatography column. 12. The method according to any one of claims 1 to 11, further comprising the steps of:
c1) cleaning the support. 13. The method according to any one of claims 1 to 12, wherein the elution of step c) comprises separating the remaining antibody free from the antibody population after contact in step b). 14. The method according to any one of claims 1 to 13, wherein the eluted antibody in step c) is a fully fucosylated antibody. 15. The method according to any one of claims 1 to 14, wherein the elution of step d) comprises contacting the support with a buffer solution which inhibits binding of the antibody to the Fc receptor. 16. The method of claim 15, wherein the buffer solution has a pH value in the range of about 3 to about 5. 17. The process according to any one of claims 1 to 16, wherein the elution of step d) is carried out at different pH values. 18. The method of claim 17, wherein the pH value comprises 4.6 and 4.2. 19. The method according to any one of claims 1 to 18, wherein the eluted antibody in step d) is a partially fucosylated and / or fully non-fucosylated antibody. 20. A method according to any one of claims 1 to 19 for analytical purposes. 21. A process according to any one of claims 1 to 20 for the purpose of manufacture. 22. The method according to any one of claims 1 to 21, further comprising the steps of:
e) collecting the eluted antibody of step c) and / or step d). 23. The method of claim 22, further comprising the steps of:
f) using the collected antibodies for experimental or therapeutic purposes. The use of an Fc receptor in a method of separating antibodies having different degrees of fucosylation. A fixed Fc receptor on a support used in a method of separating antibodies having different degrees of fucosylation. The present invention as described above.

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